Toxicity of nanosized and bulk ZnO, CuO and TiO2 to bacteria Vibrio fischeri and crustaceans Daphnia magna and Thamnocephalus platyurus

As the production of nanoparticles of ZnO, TiO2 and CuO is increasing, their (eco)toxicity to bacteria Vibrio fischeri and crustaceans Daphnia magna and Thamnocephalus platyurus was studied with a special emphasis on product formulations (nano or bulk oxides) and solubilization of particles. Our innovative approach based on the combination of traditional ecotoxicology methods and metal-specific recombinant biosensors allowed to clearly differentiate the toxic effects of metal oxides per se and solubilized metal ions. Suspensions of nano and bulk TiO2 were not toxic even at 20 g l(-1). All Zn formulations were very toxic: L(E)C50 (mg l(-1)) for bulk ZnO, nanoZnO and ZnSO4.7H2O: 1.8, 1.9, 1.1 (V. fischeri); 8.8, 3.2, 6.1 (D. magna) and 0.24, 0.18, 0.98 (T. platyurus), respectively. The toxicity was due to solubilized Zn ions as proved with recombinant Zn-sensor bacteria. Differently from Zn compounds, Cu compounds had different toxicities: L(E)C50 (mg l(-1)) for bulk CuO, nano CuO and CuSO4: 3811, 79, 1.6 (V. fischeri), 165, 3.2, 0,17 (D. magna) and 95, 2.1, 0.11 (T. platyurus), respectively. Cu-sensor bacteria showed that toxicity to V. fischeri and T. platyurus was largely explained by soluble Cu ions. However, for Daphnia magna, nano and bulk CuO proved less bioavailable than for bacterial Cu-sensor. This is the first evaluation of ZnO, CuO and TiO2 toxicity to V. fischeri and T. platyurus. For nano ZnO and nano CuO this is also a first study for D. magna.     

Toxicity of two types of silver nanoparticles to aquatic crustaceans Daphnia magna and Thamnocephalus platyurus

Although silver nanoparticles (NPs) are increasingly used in various consumer products and produced in industrial scale, information on harmful effects of nanosilver to environmentally relevant organisms is still scarce. This paper studies the adverse effects of silver NPs to two aquatic crustaceans, Daphnia magna and Thamnocephalus platyurus. For that, silver NPs were synthesized where Ag is covalently attached to poly(vinylpyrrolidone) (PVP). In parallel, the toxicity of collargol (protein-coated nanosilver) and AgNO₃ was analyzed. Both types of silver NPs were highly toxic to both crustaceans: the EC50 values in artificial freshwater were 15–17 ppb for D. magna and 20–27 ppb for T. platyurus. The natural water (five different waters with dissolved organic carbon from 5 to 35 mg C/L were studied) mitigated the toxic effect of studied silver compounds up to 8-fold compared with artificial freshwater. The toxicity of silver NPs in all test media was up to 10-fold lower than that of soluble silver salt, AgNO₃. The pattern of the toxic response of both crustacean species to the silver compounds was almost similar in artificial freshwater and in natural waters. The chronic 21-day toxicity of silver NPs to D. magna in natural water was at the part-per-billion level, and adult mortality was more sensitive toxicity test endpoint than the reproduction (the number of offspring per adult).     

Identification of a phytotoxic photo-transformation product of diclofenac using effect-directed analysis

The pharmaceutical diclofenac (DCF) is released in considerably high amounts to the aquatic environment. Photo-transformation of DCF was reported as the main degradation pathway in surface waters and was found to produce metabolites with enhanced toxicity to the green algae Scenedesmus vacuolatus. We identified and subsequently confirmed 2-[2-(chlorophenyl)amino]benzaldehyde (CPAB) as a transformation product with enhanced toxicity using effect-directed analysis. The EC₅₀ of CPAB (4.8 mg/L) was a factor of 10 lower than that for DCF (48.1 mg/L), due to the higher hydrophobicity of CPAB (log K_ow = 3.62) compared with DCF (log D_ow = 2.04) at pH 7.0.     

Copper oxide nanoparticles can induce toxicity to the freshwater shredder Allogamus ligonifer

Increased commercialisation of nanometal-based products augments the possibility of their deposition into aquatic ecosystems; this, in turn, may pose risks to aquatic biota and associated ecological functions. Freshwater invertebrate shredders mostly use microbially-colonized plant litter as food resource and play an important role in aquatic detritus food webs. We assessed lethal effects of nanoCuO on the shredder Allogamus ligonifer (Trichoptera, Limnephilidae) by determining the concentration that induced 50% of death (LC₅₀), and sublethal effects of nanoCuO on the feeding behaviour and growth of the shredder by exposing the animals to: (i) stream water supplemented with nanoCuO and microbially-colonized leaves, and (ii) stream water (without nanoCuO) and microbially-colonized leaves pre-exposed to nanoCuO. Results from acute lethal tests showed that the 96 h LC₅₀ of nanoCuO was very high (569 mg L⁻¹). In the absence of nanoparticles, leaf consumption rate was 0.27 mg leaf DM mg⁻¹ animal DM d⁻¹ and the shredder growth rate was 56 μg animal DM mg⁻¹ animal DM d⁻¹. A significant inhibition in leaf consumption rate (up to 47%) and invertebrate growth rate (up to 46%) was observed when shredders were exposed to the higher tested sublethal concentration of nanoCuO (75 mg L⁻¹) through either contaminated stream water or pre-contaminated food. The exposure to increased nanoCuO concentration via water or pre-contaminated food led to higher accumulation of copper in the larval body. Leached water-soluble ionic copper from the nanoCuO adsorbed or accumulated in the shredder (up to 10.2% of total Cu) seemed to influence the feeding behaviour and growth of the shredder.     

Use of Co speciation and soil properties to explain variation in Co toxicity to root growth of barley (Hordeum vulgare L.) in different soils

The influence of soil properties on the bioavailability and toxicity of Co to barley (Hordeum vulgare L.) root elongation was investigated. Ten soils varying widely in soil properties were amended with seven doses of CoCl₂. Soil properties greatly influenced the expression of Co toxicity. The effective concentration of added Co causing 50% inhibition (EC₅₀) ranged from 45 to 863 mg kg⁻¹, representing almost 20-fold variation among soils. Furthermore, we investigated Co toxicity in relation to Co concentrations and free Co²⁺ activity in soil solution. The EC₅₀ values showed variation among soils of 17- and 29-fold, based on the Co concentration in soil solution and free Co²⁺ activity, respectively. Single regressions were carried out between Co toxicity threshold values and selected soil properties. Models obtained showed that soil effective cation exchange capacity (eCEC) and exchangeable calcium were the most consistent single predictors of the EC₅₀ values based on soil added Co.     

Chronic toxicity of ZnO nanoparticles, non-nano ZnO and ZnCl2 to Folsomia candida (Collembola) in relation to bioavailability in soil

The chronic toxicity of zinc oxide nanoparticles (ZnO-NP) to Folsomia candida was determined in natural soil. To unravel the contribution of particle size and free zinc to NP toxicity, non-nano ZnO and ZnCl₂ were also tested. Zinc concentrations in pore water increased with increasing soil concentrations, with Freundlich sorption constants K_f of 61.7, 106 and 96.4 l/kg (n = 1.50, 1.34 and 0.42) for ZnO-NP, non-nano ZnO and ZnCl₂ respectively. Survival of F. candida was not affected by ZnO-NP and non-nano ZnO at concentrations up to 6400 mg Zn/kg d.w. Reproduction was dose-dependently reduced with 28-d EC50s of 1964, 1591 and 298 mg Zn/kg d.w. for ZnO-NP, non-nano ZnO and ZnCl₂, respectively. The difference in EC50s based on measured pore water concentrations was small (7.94-16.8 mg Zn/l). We conclude that zinc ions released from NP determine the observed toxic effects rather than ZnO particle size.     

Predicting molybdenum toxicity to higher plants: Estimation of toxicity threshold values

Four plant species (oilseed rape, Brassica napus L.; red clover, Trifolium pratense L.; ryegrass, Lolium perenne L.; and tomato, Lycopersicon esculentum L.) were tested on ten soils varying widely in soil properties to assess molybdenum (Mo) toxicity. A larger range (66-fold-609-fold) of added Mo concentrations resulting in 50% inhibition of yield (ED₅₀) was found among soils than among plant species (2-fold-38-fold), which illustrated that the soils differed widely in the expression of Mo toxicity. Toxicity thresholds based on soil solution Mo narrowed the variation among soils compared to thresholds based on added Mo concentrations. We conclude that plant bioavailability of Mo in soil depends on Mo solubility, but this alone did not decrease the variability in observed toxicity enough to be used in risk assessment and that other soil properties influencing Mo toxicity to plants need to be considered.     

Bioavailability of residual polycyclic aromatic hydrocarbons following enhanced natural attenuation of creosote-contaminated soil

The impact of residual PAHs (2250 ± 71 μg total PAHs g⁻¹) following enhanced natural attenuation (ENA) of creosote-contaminated soil (7767 ± 1286 μg total PAHs g⁻¹) was assessed using a variety of ecological assays. Microtox™ results for aqueous soil extracts indicated that there was no significant difference in EC₅₀ values for uncontaminated, pre- and post-remediated soil. However, in studies conducted with Eisenia fetida, PAH bioaccumulation was reduced by up to 6.5-fold as a result of ENA. Similarly, Beta vulgaris L. biomass yields were increased 2.1-fold following ENA of creosote-contaminated soil. While earthworm and plant assays indicated that PAH bioavailability was reduced following ENA, the residual PAH fraction still exerted toxicological impacts on both receptors. Results from this study highlight that residual PAHs following ENA (presumably non-bioavailable to bioremediation) may still be bioavailable to important receptor organisms such as earthworms and plants.     

Acute and chronic toxicity of Roundup Weathermax and Ignite 280 SL to larval Spea multiplicata and S. bombifrons from the Southern High Plains, USA

Pesticides have been implicated in widespread amphibian declines. We assessed acute and chronic toxicity of two widely used herbicides to larval New Mexico (Spea multiplicata) and Plains (S. bombifrons) spadefoots from cropland and native grassland playas. Roundup WeatherMAX^® (WM) toxicity estimates (48- and 216-h LC₅₀; 48-h LC₁) for both species were similar to environmental concentrations expected from accidental overspray. Chronic (30-day) exposure to WM at predicted environmental concentrations (2.0 and 2.8 mg glyphosate acid equivalents/L) reduced survival of both species. Ignite^® 280 SL (IG) toxicity estimates (48-h LC₅₀ and LC₁) for both species were above predicted environmental concentrations of 1.0 mg glufosinate/L. Chronic exposure to predicted environmental concentrations of IG did not reduce survival of either species. Toxicity test results suggest that at predicted environmental concentrations IG would not cause extensive mortalities among larval New Mexico and Plains spadefoots. However, WM may cause extensive mortality among larvae of these species.     

Bacterial toxicity comparison between nano- and micro-scaled oxide particles

Toxicity of nano-scaled aluminum, silicon, titanium and zinc oxides to bacteria (Bacillus subtilis, Escherichia coli and Pseudomonas fluorescens) was examined and compared to that of their respective bulk (micro-scaled) counterparts. All nanoparticles but titanium oxide showed higher toxicity (at 20 mg/L) than their bulk counterparts. Toxicity of released metal ions was differentiated from that of the oxide particles. ZnO was the most toxic among the three nanoparticles, causing 100% mortality to the three tested bacteria. Al₂O₃ nanoparticles had a mortality rate of 57% to B. subtilis, 36% to E. coli, and 70% to P. fuorescens. SiO₂ nanoparticles killed 40% of B. subtilis, 58% of E. coli, and 70% of P. fluorescens. TEM images showed attachment of nanoparticles to the bacteria, suggesting that the toxicity was affected by bacterial attachment. Bacterial responses to nanoparticles were different from their bulk counterparts; hence nanoparticle toxicity mechanisms need to be studied thoroughly.     

Comparison of the cytotoxic responses of Escherichia coli (E. coli) AMC 198 to different fullerene suspensions (nC60)

Fullerenes are set to be produced on an industrial scale in anticipation of their wide applications. This calls for research on their environmental and health impacts. This study investigates and compares the cell toxicity of different aqueous fullerene aggregates. Popular C₆₀ dispersal methods were used to prepare four types of nC₆₀ aggregates. These aggregates were tested against the indicator species Escherichia coli (E. coli) AMC 198. With aggregates of around 150 nm in diameter, the THF/nC₆₀ suspension was very toxic and gave rise to a half maximal effective concentration (EC₅₀) of 0.54 mg L⁻¹ in E. coli. By contrast, the Tol/nC₆₀ suspension exhibited a cytoprotective role while the Aqu-N₂/nC₆₀ and Aqu-O₂/nC₆₀ suspensions enhanced the metabolism of E. coli. Although some toxicants, such as THF and THF-peroxide, were introduced into the THF/nC₆₀ suspension during the dispersion, the toxicity of nC₆₀ itself cannot be neglected.     

Aggregation and ecotoxicity of CeO₂ nanoparticles in synthetic and natural waters with variable pH, organic matter concentration and ionic strength

The influence of pH (6.0-9.0), natural organic matter (NOM) (0-10 mg C/L) and ionic strength (IS) (1.7-40 mM) on 14 nm CeO₂ NP aggregation and ecotoxicity towards the alga Pseudokirchneriella subcapitata was assessed following a central composite design. Mean NP aggregate sizes ranged between 200 and 10000 nm. Increasing pH and IS enhanced aggregation, while increasing NOM decreased mean aggregate sizes. The 48 h-E_rC20s ranged between 4.7 and 395.8 mg CeO₂/L. An equation for predicting the 48 h-E_rC20 (48 h-E_rC20 = −1626.4 × (pH) + 109.45 × (pH)² + 116.49 × ([NOM]) − 14.317 × (pH) × ([NOM]) + 6007.2) was developed. In a validation study with natural waters the predicted 48 h-E_rC20 was a factor 1.08-2.57 lower compared to the experimental values.     

Acute toxicity of 353-nonylphenol and its metabolites for zebrafish embryos

Background, aim and scope  Nonylphenol (NP) can be detected in the aquatic environment all over the world. It is applied as a technical mixture of isomers of which 353-NP is the most relevant both in terms of abundance (about 20% of total mass) and endocrine potential. 353-NP is metabolised in sewage sludge. The aims of the present study were to determine and to compare the acute toxicity of t-NP, 353-NP and its metabolites as well as to discuss if the toxicity of 353-NP changes during degradation. Materials and methods  353-NP and two of its metabolites were synthesised. The zebrafish embryo test was performed according to standard protocols. Several lethal and non-lethal endpoints during embryonal development were reported. NOEL, LOEL and EC₅₀ were calculated. Results  All tested compounds caused lethal as well as non-lethal malformations during embryo development. 353-NP showed a higher toxicity (EC₅₀ for lethal endpoints 6.7 mg/L) compared to its metabolites 4-(3.5-dimethyl-3-heptyl)-2-nitrophenol (EC₅₀ 13.3 mg/L) and 4-(3,5-dimethyl-3-heptyl)-2-bromophenol (EC₅₀ 27.1 mg/L). Discussion  In surface water, concentrations of NP are far below the NOEC identified by the zebrafish embryo test. However, in soils and sewage sludge, concentrations may reach or even exceed these concentrations. Therefore, sludge-treated sites close to surface waters should be analysed for NP and its metabolites in order to detect an unduly high contamination due to runoff events. Conclusions  The results of the present study point out that the toxicity of 353-NP probably declines during metabolisation in water, sediment and soil, but does not vanish since the major metabolites exhibit a clear toxic potential for zebrafish embryos. Recommendations and perspectives  Metabolites of environmental pollutants should be included in the ecotoxicological test strategy for a proper risk assessment. An erratum to this article can be found at     

Temporal genetic variability and host sources of Escherichia coli associated with fecal pollution from domesticated animals in the shellfish culture environment of Xiangshan Bay, East China Sea

This study was conducted to analyze the genetic variability of Escherichia coli from domesticated animal wastes for microbial source tracking (MST) application in fecal contaminated shellfish growing waters of Xiangshan Bay, East China Sea. (GTG)₅ primer was used to generate 1363 fingerprints from E. coli isolated from feces of known 9 domesticated animal sources around this shellfish culture area. Jackknife analysis of the complete (GTG)₅-PCR DNA fingerprint library indicated that isolates were assigned to the correct source groups with an 84.28% average rate of correct classification. Based on one-year source tracking data, the dominant sources of E. coli were swine, chickens, ducks and cows in this water area. Moreover, annual and spatial changes of E. coli concentrations and host sources may affect the level and distribution of zoonotic pathogen species in waters. Our findings will further contribute to preventing fecal pollution in aquatic environments and quality control of shellfish.     

Heterogeneous loss of HO2 by KCl,synthetic sea salt,and natural seawater aerosol particles

The HO₂ uptake to aerosol particles is potentially significant sink for the HO₂ radical in the marine atmosphere. To assess the heterogeneous loss of HO₂ on marine aerosol particles, we have investigated the uptake coefficients (γ) of HO₂ for submicron aerosol particles of KCl, synthetic sea salt, and natural seawater under ambient conditions (760 Torr and 296 ± 2 K) using an aerosol flow tube (AFT) coupled with a chemical conversion/laser-induced fluorescence (CC/LIF) technique. γ values determined for dry and wet aerosols of KCl were 0.02 ± 0.01 and 0.07 ± 0.03 at 66% and 75% RH, respectively, while γ values for those doped with CuSO₄ was 0.55 ± 0.19 at 75% RH. γ values determined for synthetic sea-salt particles were 0.07 ± 0.03, 0.12 ± 0.04 and 0.13 ± 0.04 at 35%, 50%, 75% RH, respectively, while γ values for natural seawater particles were 0.10 ± 0.03, 0.11 ± 0.02 and 0.10 ± 0.03 at 35%, 50%, 75% RH, respectively. We recommend a HO₂ uptake coefficient in marine areas of 0.1 for modeling and estimated the contribution of heterogeneous loss of HO₂ by sea-salt aerosol particles in marine areas using a box model. Our box-model simulations suggested that daytime maximum HO₂ concentrations decreased to 87–94% of the values without heterogeneous loss.     

Joint action and lethal levels of toluene, ethylbenzene, and xylene on midge (Chironomus plumosus) larvae

Aquatic ecosystems are vulnerable to the exposure with petrochemicals such as toluene, ethylbenzene, and xylene (o-, m-, and p-xylene) (TEX) and their adverse effects. Considering the widespread use, occurrence, and high toxicity of TEX, the aim of this work was to investigate the differential toxicity of TEX against midge (Chironomus plumosus) larvae and reveal the joint action of binary and ternary mixtures of TEX using the predictive concentration addition model. More importantly, this research can afford the basic toxicity data and scientific reference for the establishment of water quality criteria or benchmark, water pollution control, and aquatic risk assessment. Single and joint toxic effects of TEX on C. plumosus larvae were investigated using a semi-static bioassay, and the type of joint effects of TEX was ascertained. In the single toxicant experiments, the toxicity of the three pollutants could be sequenced as ethylbenzene > xylene > toluene. Specifically, LC_50s of T, E, and X after a 48-h exposure were 64.9, 37.8, and 42.0 mg/L, respectively. In the binary mixture experiments, the interaction between toluene and ethylbenzene, ethylbenzene and xylene, and toluene and xylene was largely in conformity with partial additive or additive effect as determined by isobologram representation and toxic unit models. In the ternary mixture experiments, the interaction was basically dependent on the use of additive index and mixture toxicity index methods. However, the antagonistic and synergistic actions were not significant. Thus, the tertiary mixture interaction could be regarded as additive action. The concentration addition model could successfully predict the joint action of TEX mixtures on C. plumosus larvae. Particularly, the additive action of TEX on C. plumosus larvae can be further recommended to evaluate water quality criteria of TEX.     

Sandhopper solar orientation as a behavioural biomarker of trace metals contamination

Although many studies have focused on trace metals accumulation, investigations of talitrid amphipods as biomarkers are rare. This study explores the possibility of using the solar orientation capacity of Talitrus saltator as a behavioural marker of exposure to two essential (Cu and Zn) and two non-essential (Cd and Hg) metals. LC₅₀ analyses performed before the solar orientation tests showed that the 72 h LC₅₀ for Hg was 0.02 ppm while the 96 h LC₅₀ values for Cu, Cd and Zn were 13.28 ppm, 27.66 ppm, and 62.74 ppm, respectively. The presence of metals in seawater affects the solar orientation capacity of T. saltator in a concentration-dependent manner and according to the toxicity ranking of the metals (Hg > Cu > Cd > Zn). Therefore, the solar orientation capacity of T. saltator seems to be a promising behavioural marker for exposure to trace metals.     

Toxicity of nanoparticulate and bulk ZnO, Al2O3 and TiO2 to the nematode Caenorhabditis elegans

Limited information is available on the environmental behavior and associated potential risk of manufactured oxide nanoparticles (NPs). In this research, toxicity of nanoparticulate and bulk ZnO, Al₂O₃ and TiO₂ were examined to the nematode Caenorhabditis elegans with Escherichia coli as a food source. Parallel experiments with dissolved metal ions from NPs were also conducted. The 24-h median lethal concentration (LC₅₀) and sublethal endpoints were assessed. Both NPs and their bulk counterparts were toxic, inhibiting growth and especially the reproductive capability of the nematode. The 24-h LC₅₀ for ZnO NPs (2.3 mg L⁻¹) and bulk ZnO was not significantly different, but significantly different between Al₂O₃ NPs (82 mg L⁻¹) and bulk Al₂O₃ (153 mg L⁻¹), and between TiO₂ NPs (80 mg L⁻¹) and bulk TiO₂ (136 mg L⁻¹). Oxide solubility influenced the toxicity of ZnO and Al₂O₃ NPs, but nanoparticle-dependent toxicity was indeed observed for the investigated NPs.     

Toxicity evaluation of cypermethrin,glyphosate, and malathion,on two indigenous zooplanktonic species

In Aguascalientes, Mexico, there is a special concern about pesticides because of their intensive use on guava production areas, which are located in the vicinity of water reservoirs; thus, non-target organisms could be exposed. Thereafter, the aim of this work was to assess the effect of cypermethrin, Faena® (glyphosate), and malathion, which are the most used pesticides in Aguascalientes’ guava production, on the indigenous freshwater species Alona guttata (cladoceran) and Lecane papuana (rotifer). Acute 48-h toxicity tests were carried out, and LC₅₀ values were calculated. Then, five sublethal concentrations (1/80, 1/40, 1/20, 1/10, and 1/5 of the respective LC₅₀) were selected for the chronic assays: (a) intrinsic growth rate analysis in the rotifer and (b) partial life table analysis in the cladoceran. The results of the acute toxicity tests showed that A. guttata was more sensitive to malathion (LC₅₀ = 5.26 × 10^?3 mg/L) at concentrations found in natural environments with continuous application on guava fields, whereas L. papuana was more sensitive to Faena® (LC₅₀ = 19.89 mg/L). The somatic growth of A. guttata was inhibited for the chronic exposure to cypermethrin. In addition, cypermethrin and Faena® seemed to exert endocrine disruptive effects on A. guttata. Moreover, malathion chronic exposure significantly decreased the survival of A. guttata. Moreover, L. papuana was affected chronically for the three pesticides.     

Behavioural and biochemical responses of two marine invertebrates Scrobicularia plana and Hediste diversicolor to copper oxide nanoparticles

Engineered nano-sized Cu oxide particles are extensively used in diverse applications. Because aquatic environments are the ultimate “sink” for all contaminants, it is expected that nanoparticles (NP) will follow the same fate. In this study, two marine invertebrates Scrobicularia plana and Hediste diversicolor were chosen as ecotoxicological models. The aim was to evaluate behavioural (burrowing kinetics, feeding rate) and biochemical (biomarkers) responses of S. plana and H. diversicolor exposed in the laboratory to Cu (10 μg L⁻¹) added in natural seawater either in the form of engineered nanoparticles (NPs) of CuO or as dissolved Cu in 2% HNO₃. Exposure was characterized by considering (i) the physico-chemical fate of NP (ii) the fraction of labile Cu in experimental media and (iii) Cu bioaccumulation. Results showed high aggregation of CuO NPs in seawater and no additional bioavailable Cu concentrations. Behavioural impairments were observed in S. plana exposed to CuO NPs or soluble Cu whereas in H. diversicolor, only the exposure to soluble Cu led to a burrowing decrease. No obvious neurotoxicity effects were revealed since in both species, no changes in cholinesterasic activity occurred in response to both forms of Cu exposure. Biomarkers of oxidative-stress catalase and glutathione-S-transferase were enhanced in both species whereas superoxide dismutase was increased only in S. plana exposed to CuO NPs. Metallothionein-like protein was increased in bivalves exposed to both forms of Cu. Since, no detectable release of soluble Cu from CuO NPs occurred during the time of experiment, ecotoxicity effects seem to be related to CuO NPs themselves.